Alginate is a copolymer of 1,4-linked .beta.-D-mannuronic and .alpha.-L-guluronic acid. It has the unique property of gel-formation in the presence of divalent actions such as calcium, and has been used to immobilize cells (M.F.A. Goosen et al., "Microencapsulation of Living Tissue and Cells," Canadian Patent 1,215,922 (1982)), as potential artificial organs (M.F.A. Goosen et al., "Optimization of Microencapsulation Parameters: Semipermeable Microcapsules as a Bioartificial Pancreas," Biotech. Bioeng. 27:146-150 (1985)), and as delivery systems for drugs (C.K. Kim and E.J. Lee, "The Controlled Release of Blue Dextran from Alginate Beads," Int. d. Pharm. 79:11-19 (1992)), pesticides (A.B. Pepperman et al., "Alginate Controlled Release Formulations of Metribuzin," J. Cont. Rel. 17:105-112 (1991)), and herbicides (G. Pfisteret al., "Release Characteristics of Herbicides from Ca Alginate Gel Formulations," J. Cont. Rel. 3:229-233 (1986)). The gelation and crosslinking of alginate with calcium are due to the stacking of guluronic acid (G) blocks with the formation of `egg-box` junctions (A. Katchalsky et al., "Counter-Ion Fixation in Alginates," J. Chem. Soc. :5198-5204 (1961)). Thus, the dropwise addition of an alginate solution into a stirred solution of calcium chloride and drug will result in the encapsulation of the drug within an alginate hydrogel bead containing up to 95% water (M.F.A. Goosen et al., "Microencapsulation of Living Tissue and Cells," Canadian Patent 1,215,922 (1982)).
Purified alginate is non-toxic when taken orally, biodegradable and bioacceptable (C.K. Kim and E.J. Lee, "The Controlled Release of Blue Dextran from Alginate Beads," Int. J. Pharm. 79:11-19 (1992); O.N. Singh and D.J. Burgess, "Characterization of Albumin-Alginic Acid Complex Coacervation," J. Pharm. Pharmacol. 41:670-673 (1989)). Alginate has also been tested clinically as a component of synthetic pancreatic beta cells (as artificial pancreatic material). In addition, alginate has been found to have a protective effect on the mucous membranes of the upper gastrointestinal tract (D. Koji etal., "Pharmacological Studies of Sodium Alginate. I. Protective Effect of Sodium Alginate on Mucous Membranes of Upper-Gastrointestinal Tract," Yakugaku Zasshi 101:452-457 (1981)) and has been investigated as an bioadhesive (D. Chickering et al., "A Tensile Technique to Evaluate the Interaction of Bioadhesive Microspheres with Intestinal Mucosa," Proc. Int. Symp. Cont. Rel. Bio. Mater. 19:88-89 (1992)). Alginate beads have been used as a potential delivery system for many cationic drugs such as propranolol (N. Segi et al., "Interaction of Calcium-Induced Alginate Gel Beads with Propranolol," Chem. Pharm. Bull. 37:3092-3095 (1989)) chlorpheniramine (A.F. Stockwell et al., "In Vitro Evaluation of Alginate Gel Systems as Sustained Release Drug Delivery Systems," J. Contr. Rel. 3:167-175 (1986)), theophylline (M. Bhakoo et al., "Release of Antibiotics and Antitumour Agents from Alginate and Gellan Gum Gels," Proc. Int. Symp. Cont. Rel. Bio. Mater. 18:441-442 (1991)), and protein growth factors such as basic fibroblast growth factor (bFGF) (E.C. Downsetal., "Calcium Alginate Beads as a Slow-Release System for Delivering Angiogenic Molecules In Vivo and In Vitro," J. Cell. Phys. 152:422-429 (1992), E.R. Edelman et al., "Controlled and Modulated Release of Basic Fibroblast Growth Factor," Biomaterials 12:619-625 (1991)). The mucosal epithelium of the intestine is in a continuously dynamic state known as "epithelial renewal" (G.L. Eastwood, Gastroenterology 72:962 (1980)) where undifferentiated stem cells from the proliferative crypt zone divide, differentiate and migrate to the luminal surface where, once terminally differentiated, are sloughed from the tips of the villi. The turnover of a crypt-villus cell population is rapid and occurs every 24-72 h (H. Cheng and C. Leblond, Am. J. Anat. 141:461 (1974)). Continuous exfoliation of the cells at the viiius tip is counterbalanced by ongoing proliferation in the crypt so that the net intestinal epithelial mass remains relatively constant. The multifactorial regulation of this balance is not fully understood (Physiology of the Gastrointestinal Tract, L.R. Johnson, Ed. (Raven Press, New York), pp. 69-196 (1987)). It may, however, be accomplished through the combined integration of key peptide growth factors and constituents of the extracellular matrix (D.K Podolsky, Am. J. Physiol. 264:G1.79 (1993)). Transforming growth factor beta (TGF-.beta.), an acid and heat stable, disulfide-linked, homodimeric 25 kD protein is present in most tissues and is known to play an important regulatory role in cell proliferation, migration, and differentiation (R.K. Assoian et al., J. Biol. Chem. 258:7155 (1983); M.B. Spornet al., Science 233:532 (1986); J. Massague, Cell 49:437 (1987)). TGF-.beta. has been shown to inhibit the growth of many cells of epithelial origin, including human and rodent derived intestinal cells (M. Kurokawa et al., Biochem. Biophys. Res. Comm. 142:775 (1987) and J.A. Barnard et al., Proc. Natl. Acad. Sci. USA 86:1578 (1989)). TGF-.beta. mRNA is expressed in gastrointestinal epithelium (R.P. McCabe et al., Clin. Immunol. Immunopathol. 66:52 (1993)) and the non-transformed rat jejunal crypt cell line (IEC-6) express TGF-.beta.mRNA (S. Koyama and D.K. Podolsky, J. Clin. Invest. 83:1768 (1989)) and secretes latent TGF-.beta. (M. Kurokawa et al., Biochem. Biophys. Res. Comm. 142:775 (1987) and J.A. Barnard et at., Proc. Natl. Acad. Sci. USA 86:1578 (1989)).
The rapidly proliferating epithelium of the gastrointestinal tract is extremely sensitive to cytotoxic drugs widely used in chemotherapy of cancer. The tolerable dose of these drugs is limited and often suboptimal dosages have to be used because of gut toxicity. Trials in cancer patients receiving chemotherapeutic agents have demonstrated a variety of gastrointestinal complications ranging from dyspepsia to life threatening hemorrhage from mucosal ulcerations. As many as 50% of lung cancer patients enrolled in a cisplatin plus etoposide combination chemotherapy trial were unable to complete the treatment protocol because of excessive acute gastrointestinal toxicity (S. Sartori etal., Oncology 48:356 (1991)). Sequential chemotherapeutic protocols using cytosine arabinoside, floxuridine and mitomycinC induced gastrointestinal toxic alterations characterized by surface and glandular epithelial atypia, immaturity and necrosis (R.E. Slavin et al., Cancer 42:1747 (1978)) often leading to severe systemic infections by streptococci, candida and other pathogens.
Certain growth factors exhibit gastroprotective activities and enhance the healing of gastric lesions (S.J. Konturek et al., Scand. J. Gastroenterol. 27:649 (1992)). Since TGF-.beta. inhibits the proliferation of intestinal epithelial cells, it would be highly desirable to have access to a suitable system for the oral delivery of TGF-.beta..sub.1 to the gastrointestinal tract in active forms and/or for the delivery of TGF-.beta..sub.1 or other cationic drugs to other delivery target areas. Due to its nontoxic nature, an alginate-based delivery system would appear useful for this purpose.
However, for encapsulation of cationic drugs, such as TGF-.beta..sub.1, strong complexation between alginate and the cationic drug leads to increased drug loadings into beads but decreased diffusion rates out of beads. In addition, several authors observed competition between calcium ions and the cationic drug for available carboxylic acid sights on alginate (N. Segi et al., "Interaction of Calcium-Induced Alginate Gel Beads with Propranolol," Chem. Pharm. Bull. 37:3092-3095 (1989); A.F. Stockwell et al., "In Vitro Evaluation of Alginate Gel Systems as Sustained Release Drug Delivery Systems," J. Contr. Rel. 3:167-175 (1986)).
Thus, while alginate beads have been known in the an as potential delivery systems for various therapeutic drugs, it has been found that complex interaction between alginate and cationic therapeutic agents prevents release of the therapeutic agents from the alginate beads in active form. Accordingly, there is a strong need in the art for improved alginate delivery systems for cationic therapeutic agents.